靶向hTERT的RNAi载体的构建及其对大肠癌SW-480细胞增殖的影响  

Effect of RNAi-mediated silencing of the human telomerase reverse transcriptase gene on colorectal cancer cell proliferation in vitro

作　　者：孙彦[1] 孟永亮[1] 

机构地区：[1]山东万杰医学院医学系内科学教研室,山东淄博255213

出　　处：《中国肿瘤生物治疗杂志》2014年第4期423-427,共5页Chinese Journal of Cancer Biotherapy

基　　金：山东万杰医学院校级课题(No.X10ZK02)~~

摘　　要：目的:构建靶向人大肠癌细胞端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)基因的RNAi载体,探讨其对人大肠癌SW480细胞增殖的影响。方法:设计3条靶向hTERT的shRNA序列和阴性对照序列,分别克隆入pGPU6/GFP/Neo载体,构建RNAi载体pGPU6-hTERT-1、2、3和阴性对照载体pGPU6-hTERT-NC,转染SW480细胞。RT-PCR检测各组载体对hTERT mRNA表达的影响,MTT法检测下调hTERT mRNA表达对SW480细胞增殖的影响。结果:成功构建3个携带hTERT mRNA序列的重组载体,3种RNAi载体均能明显抑制SW480细胞hTERT mRNA的表达,pGPU6-hTERT-3组SW480细胞hTERT mRNA表达水平较空白对照组下调最为显著(0.347±0.028 vs 0.513±0.032,P<0.01)。转染3种RNAi载体均能明显抑制SW480细胞增殖,pGPU6-hTERT-3组细胞增殖抑制率较空白对照组、脂质体对照组和pGPU6-hTERT-NC组升高最为显著[(50.08±0.43)%vs(4.11±0.39)%、(3.88±0.35)%、(3.38±0.35)%,P<0.05]。结论:转染RNAi载体pGPU6-hTERT-3能够抑制SW480细胞的增殖,其机制可能与降低hTERT基因的表达从而抑制端粒酶活性有关。Objective： To construct an optimized human telomerase reverse transcriptase（ hTERT） gene-specific RNAi and to evaluate its effect on human colon cancer cell proliferation in vitro. Method： Three hTERT-specific RNAi sequences and a negative control（ NC） or scrambled sequence were cloned,respectively,into a pGPU6 /GFP /Neo vector to generate pGPU6-GFP-hTERT-1,pGPU6-GFP-hTERT-2,pGPU6-GFP-hTERt-3 and pGPU6-GFP-NC. Human colon cancer SW480 cells were transfected with these vectors respectively. At 24,48 and 72 h after transfection,hTERT mRNA abundance was assessed by RT-PCR and cell viability by MTT assay. Results： The 3 hTERT-specific RNAi vectors constructed were all effective to silence the hTERT gene; hTERT mRNA abundance in SW480 cells transfected with pGPU6-GFP-hTERT-3 was significantly lower than that in SW480 cells transfected with pGPU6-GFP-NC（ 0. 347 ± 0. 028 vs0. 513 ± 0. 032,P 0. 01）. All the three hTERT sequence-specific RNAi vectors were effective to inhibit the proliferation of SW480 cells; cellular proliferation inhibition rate in SW480 cells of pGPU6-GFP-hTERT-3 group was significantly increased than that of blank contro,liposomal and NC group（ [50. 08 ± 0. 43]% vs [4. 11 ± 0. 39]%,[3. 88 ±0. 35]% and [3. 38 ± 0. 35]%; P 0. 05）. Conclusion： RNAi-mediated hTERT gene silencing results in colon cancer cell growth inhibition and may offer a novel therapy for colon cancer.

关 键 词：大肠癌 RNA干扰 人端粒酶逆转录酶 SW-480细胞 增殖 

分 类 号：R735.3[医药卫生—肿瘤] R730.54[医药卫生—临床医学]